Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques

Meishen Ren; Hong Mei; Ming Zhou; Zhen F. Fu; Heyou Han; Dingren Bi; Fuhu Peng; Ling Zhao

doi:10.1007/s12250-020-00323-1

April 2021

Meishen Ren, Hong Mei, Ming Zhou, Zhen F. Fu, Heyou Han, Dingren Bi, Fuhu Peng and Ling Zhao. Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques[J]. Virologica Sinica, 2021, 36(2): 220-230. doi: 10.1007/s12250-020-00323-1

Citation: Meishen Ren, Hong Mei, Ming Zhou, Zhen F. Fu, Heyou Han, Dingren Bi, Fuhu Peng, Ling Zhao. Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques .VIROLOGICA SINICA, 2021, 36(2) : 220-230. http://dx.doi.org/10.1007/s12250-020-00323-1

通过CRISPR技术开发高灵敏度的非洲猪瘟检测方法

任梅渗 ^1,2 ,
梅红 ^1,2 ,
周明 ^1,2 ,
傅振芳 ^1,2 ,
韩鹤友 ^1,3 ,
毕丁仁 ^1,2 ,
彭伏虎 ⁴ ,
赵凌 ^1,2,,

cstr: 32224.14.s12250-020-00323-1

1.
农业微生物国家重点实验室
2.
湖北省预防兽医学重点实验室
3.
华中农业大学动科动医学院

通讯作者： 赵凌, lingzhao@mail.hzau.edu.cn, ORCID: http://orcid.org/0000-0003-0569-8105
收稿日期： 2020-05-12
录用日期： 2020-10-20
出版日期： 2021-01-07

摘要

非洲猪瘟（ASF）是一种由非洲猪瘟病毒（ASFV）引起的传染病，其临床症状为高烧，大出血和高死亡率，对全球猪业和粮食安全构成威胁。对ASFV的隔离和控制对于防止养猪业受到ASFV损害至关重要。这项研究中开发了一种基于重组酶聚合酶扩增（RPA）-CRISPR的核酸检测方法来诊断ASF。作为一种高度灵敏的方法，RPA-CRISPR可以通过定量实时PCR（qPCR）检测通过CRISPR-lwCas13a（也称为C2c2）的并行切割引起的荧光信号，该方法甚至可以检测到低至单个拷贝的ASFV质粒和基因组DNA，并且具有比传统qPCR方法相同甚至更高的灵敏度。本研究也开发了一种试纸条与RPA-CRISPR结合用于ASFV检测，具有与TaqMan qPCR相同的灵敏度。同样，RPA-CRISPR能够将ASFV基因组DNA与其他猪病病毒的DNA / RNA区别开，而没有任何交叉反应性。此诊断方法也可用于诊断ASFV临床DNA样品，对于ASFV阳性和阴性样品的符合率均为100％。作为一种高度灵敏的检测方法，RPA-CRISPR在养猪业和食品安全领域均具有对ASFV进行临床检疫的巨大潜力。
- 非洲猪瘟（ASFV）
- , 重组酶聚合酶扩增（RPA）
- , CRISPR-lwCas13a
- , 高灵敏检测

Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques

Meishen Ren ^1,2 ,
Hong Mei ^1,2 ,
Ming Zhou ^1,2 ,
Zhen F. Fu ^1,2 ,
Heyou Han ^1,3 ,
Dingren Bi ^1,2 ,
Fuhu Peng ⁴ ,
Ling Zhao ^1,2,,

1.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
2.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
3.
College of Science, Huazhong Agricultural University, Wuhan 430070, China
4.
Hubei Center for Animal Disease Control and Prevention, Wuhan 430070, China

Corresponding author: Ling Zhao, lingzhao@mail.hzau.edu.cn
ORCID: http://orcid.org/0000-0003-0569-8105
Received Date: 12 May 2020
Accepted Date: 20 October 2020
Published Date: 07 January 2021

Abstract

African swine fever (ASF) is an infectious disease caused by African swine fever virus (ASFV) with clinical symptoms of high fever, hemorrhages and high mortality rate, posing a threat to the global swine industry and food security. Quarantine and control of ASFV is crucial for preventing swine industry from ASFV infection. In this study, a recombinase polymerase amplification (RPA)-CRISPR-based nucleic acid detection method was developed for diagnosing ASF. As a highly sensitive method, RPA-CRISPR can detect even a single copy of ASFV plasmid and genomic DNA by determining fluorescence signal induced by collateral cleavage of CRISPR-lwCas13a (previously known as C2c2) through quantitative real-time PCR (qPCR) and has the same or even higher sensitivity than the traditional qPCR method. A lateral flow strip was developed and used in combination with RPA-CRISPR for ASFV detection with the same level of sensitivity of TaqMan qPCR. Likewise, RPA-CRISPR is capable of distinguishing ASFV genomic DNA from viral DNA/RNA of other porcine viruses without any cross-reactivity. This diagnostic method is also available for diagnosing ASFV clinical DNA samples with coincidence rate of 100% for both ASFV positive and negative samples. RPA-CRISPR has great potential for clinical quarantine of ASFV in swine industry and food security.
- African swine fever (ASFV)
- , Recombinase polymerase amplification (RPA)
- , CRISPR-lwCas13a
- , Sensitive diagnosis

References
1. Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F (2016) C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353:aaf5573
2. Aguero M, Fernandez J, Romero L, Sanchez Mascaraque C, Arias M, Sanchez-Vizcaino JM (2003) Highly sensitive PCR assay for routine diagnosis of African swine fever virus in clinical samples. J Clin Microbiol 41:4431-4434
  doi: 10.1128/JCM.41.9.4431-4434.2003
3. Ai JW, Zhou X, Xu T, Yang M, Chen Y, He GQ, Pan N, Cai Y, Li Y, Wang X, Su H, Wang T, Zeng W, Zhang WH (2019) CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis. Emerg Microbes Infect 8:1361-1369
  doi: 10.1080/22221751.2019.1664939
4. Basto AP, Portugal RS, Nix RJ, Cartaxeiro C, Boinas F, Dixon LK, Leitao A, Martins C (2006) Development of a nested PCR and its internal control for the detection of African swine fever virus (ASFV) in Ornithodoros erraticus. Arch Virol 151:819-826
  doi: 10.1007/s00705-005-0654-2
5. Chang Y, Deng Y, Li T, Wang J, Wang T, Tan F, Li X, Tian K (2019) Visual detection of porcine reproductive and respiratory syndrome virus using CRISPR-Cas13a. Transbound Emerg Dis 67:564-571
6. Chen Y, Song T, Xiao YL, Wan X, Yang L, Li J, Zeng G, Fang P, Wang ZZ, Gao R (2018) Enhancement of immune response of piglets to PCV-2 vaccine by porcine IL-2 and fusion IL-4/6 gene entrapped in chitosan nanoparticles. Res Vet Sci 117:224-232
  doi: 10.1016/j.rvsc.2017.12.004
7. Dixon LK, Sun H, Roberts H (2019) African swine fever. Antiviral Res 165:34-41
  doi: 10.1016/j.antiviral.2019.02.018
8. East-Seletsky A, O'Connell MR, Knight SC, Burstein D, Cate JH, Tjian R, Doudna JA (2016) Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature 538:270-273
  doi: 10.1038/nature19802
9. Fernandez-Pinero J, Gallardo C, Elizalde M, Robles A, Gomez C, Bishop R, Heath L, Couacy-Hymann E, Fasina FO, Pelayo V, Soler A, Arias M (2013) Molecular diagnosis of African Swine Fever by a new real-time PCR using universal probe library. Transbound Emerg Dis 60:48-58
  doi: 10.1111/j.1865-1682.2012.01317.x
10. Freuling CM, Muller TF, Mettenleiter TC (2017) Vaccines against pseudorabies virus (PrV). Vet Microbiol 206:3-9
  doi: 10.1016/j.vetmic.2016.11.019
11. Galindo I, Alonso C (2017) African Swine Fever Virus:A Review. Viruses 9:103
  doi: 10.3390/v9050103
12. Gaudreault NN, Richt JA (2019) Subunit Vaccine Approaches for African Swine Fever Virus. Vaccines 7:56
  doi: 10.3390/vaccines7020056
13. Ge S, Li J, Fan X, Liu F, Li L, Wang Q, Ren W, Bao J, Liu C, Wang H, Liu Y, Zhang Y, Xu T, Wu X, Wang Z (2018) Molecular Characterization of African Swine Fever Virus, China, 2018. Emerging Infect Dis 24:2131-2133
  doi: 10.3201/eid2411.181274
14. Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F (2018) Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a and Csm6. Science 360:439-444
  doi: 10.1126/science.aaq0179
15. Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F (2017) Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356:438-442
  doi: 10.1126/science.aam9321
16. Haines FJ, Hofmann MA, King DP, Drew TW, Crooke HR (2013) Development and validation of a multiplex, real-time RT PCR assay for the simultaneous detection of classical and African swine fever viruses. PLoS ONE 8:e71019
  doi: 10.1371/journal.pone.0071019
17. Han J, Zhou L, Ge X, Guo X, Yang H (2017) Pathogenesis and control of the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus. Vet Microbiol 209:30-47
  doi: 10.1016/j.vetmic.2017.02.020
18. Karger A, Perez-Nunez D, Urquiza J, Hinojar P, Alonso C, Freitas FB, Revilla Y, Le Potier MF, Montoya M (2019) An Update on African Swine Fever Virology. Viruses 11:864
  doi: 10.3390/v11090864
19. Kim HJ, Lee MJ, Lee SK, Kim DY, Seo SJ, Kang HE, Nam HM (2019) African Swine Fever Virus in Pork Brought into South Korea by Travelers from China, August 2018. Emerg Infect Dis 25:1231-1233
  doi: 10.3201/eid2506.181684
20. King DP, Reid SM, Hutchings GH, Grierson SS, Wilkinson PJ, Dixon LK, Bastos AD, Drew TW (2003) Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus. J Virol Methods 107:53-61
  doi: 10.1016/S0166-0934(02)00189-1
21. Liu Y, Xu H, Liu C, Peng L, Khan H, Cui L, Huang R, Wu C, Shen S, Wang S, Liang W, Li Z, Xu B, He N (2019) CRISPR-Cas13a Nanomachine Based Simple Technology for Avian Influenza A (H7N9) Virus On-Site Detection. J Biomed Nanotechnol 15:790-798
  doi: 10.1166/jbn.2019.2742
22. Luo Y, Li S, Sun Y, Qiu HJ (2014) Classical swine fever in China:a minireview. Vet Microbiol 172:1-6
  doi: 10.1016/j.vetmic.2014.04.004
23. Luo Y, Atim SA, Shao L, Ayebazibwe C, Sun Y, Liu Y, Ji S, Meng XY, Li S, Li Y, Masembe C, Stahl K, Widen F, Liu L, Qiu HJ (2017) Development of an updated PCR assay for detection of African swine fever virus. Arch Virol 162:191-199
  doi: 10.1007/s00705-016-3069-3
24. Malogolovkin A, Kolbasov D (2019) Genetic and antigenic diversity of African swine fever virus. Virus Res 271:197673
  doi: 10.1016/j.virusres.2019.197673
25. Mazur-Panasiuk N, Zmudzki J, Wozniakowski G (2019) African Swine Fever Virus-Persistence in Different Environmental Conditions and the Possibility of its Indirect Transmission. J Vet Res 63:303-310
  doi: 10.2478/jvetres-2019-0058
26. Myhrvold C, Freije CA, Gootenberg JS, Abudayyeh OO, Metsky HC, Durbin AF, Kellner MJ, Tan AL, Paul LM, Parham LA, Garcia KF, Barnes KG, Chak B, Mondini A, Nogueira ML, Isern S, Michael SF, Lorenzana I, Yozwiak NL, MacInnis BL, Bosch I, Gehrke L, Zhang F, Sabeti PC (2018) Field-deployable viral diagnostics using CRISPR-Cas13. Science 360:444-448
27. Ngom B, Guo Y, Wang X, Bi D (2010) Development and application of lateral flow test strip technology for detection of infectious agents and chemical contaminants:a review. Anal Bioanal Chem 397:1113-1135
  doi: 10.1007/s00216-010-3661-4
28. Olesen AS, Lohse L, Boklund A, Halasa T, Gallardo C, Pejsak Z, Belsham GJ, Rasmussen TB, Botner A (2017) Transmission of African swine fever virus from infected pigs by direct contact and aerosol routes. Vet Microbiol 211:92-102
  doi: 10.1016/j.vetmic.2017.10.004
29. Piepenburg O, Williams CH, Stemple DL, Armes NA (2006) DNA detection using recombination proteins. PLoS Biol 4:e204
  doi: 10.1371/journal.pbio.0040204
30. Qin P, Park M, Alfson KJ, Tamhankar M, Carrion R, Patterson JL, Griffiths A, He Q, Yildiz A, Mathies R, Du K (2019) Rapid and Fully Microfluidic Ebola Virus Detection with CRISPR-Cas13a. ACS Sens 4:1048-1054
  doi: 10.1021/acssensors.9b00239
31. Quesada-Gonzalez D, Merkoci A (2015) Nanoparticle-based lateral flow biosensors. Biosens Bioelectron 73:47-63
  doi: 10.1016/j.bios.2015.05.050
32. Sanchez EG, Perez-Nunez D, Revilla Y (2019) Development of vaccines against African swine fever virus. Virus Res 265:150-155
  doi: 10.1016/j.virusres.2019.03.022
33. Shmakov S, Smargon A, Scott D, Cox D, Pyzocha N, Yan W, Abudayyeh OO, Gootenberg JS, Makarova KS, Wolf YI, Severinov K, Zhang F, Koonin EV (2017) Diversity and evolution of class 2 CRISPR-Cas systems. Nat Rev Microbiol 15:169-182
  doi: 10.1038/nrmicro.2016.184
34. Simulundu E, Lubaba CH, van Heerden J, Kajihara M, Mataa L, Chambaro HM, Sinkala Y, Munjita SM, Munang'andu HM, Nalubamba KS, Samui K, Pandey GS, Takada A, Mweene AS (2017) The Epidemiology of African Swine Fever in "Nonendemic" Regions of Zambia (1989-2015):Implications for Disease Prevention and Control. Viruses 9:236
  doi: 10.3390/v9090236
35. Smargon AA, Cox DBT, Pyzocha NK, Zheng K, Slaymaker IM, Gootenberg JS, Abudayyeh OA, Essletzbichler P, Shmakov S, Makarova KS, Koonin EV, Zhang F (2017) Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28. Mol Cell 65:618-630
  doi: 10.1016/j.molcel.2016.12.023
36. Teklue T, Sun Y, Muhammad A, Luo Y, Qiu HJ (2019) Current status and evolving approaches to African swine fever vaccine development. Transbound Emerg Dis 67:529-542
37. Wang A, Jia R, Liu Y, Zhou J, Qi Y, Chen Y, Liu D, Zhao J, Shi H, Zhang J, Zhang G (2019) Development of a novel quantitative real-time PCR assay with lyophilized powder reagent to detect African swine fever virus in blood samples of domestic pigs in China. Transbound Emerg Dis 67:284-297
38. Wang D, Yu J, Wang Y, Zhang M, Li P, Liu M, Liu Y (2019) Development of a real-time loop-mediated isothermal amplification (LAMP) assay and visual LAMP assay for detection of African swine fever virus (ASFV). J Virol Methods 276:113775
39. Wang J, Wang J, Geng Y, Yuan W (2017) A recombinase polymerase amplification-based assay for rapid detection of African swine fever virus. Can J Vet Res 81:308-312
40. Wang T, Sun Y, Qiu HJ (2018) African swine fever:an unprecedented disaster and challenge to China. Infect Dis Poverty 7:111
  doi: 10.1186/s40249-018-0495-3
41. Zhao D, Liu R, Zhang X, Li F, Wang J, Zhang J, Liu X, Wang L, Zhang J, Wu X, Guan Y, Chen W, Wang X, He X, Bu Z (2019) Replication and virulence in pigs of the first African swine fever virus isolated in China. Emerg Microbes Infect 8:438-447
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